JP7835397B2 - Methods, apparatus, and computer programs for assisting in determining the level of vascular dysplasia. - Google Patents

Description

This specification discloses methods, apparatus, and computer programs for assisting in determining the level of vascular dysplasia in the gastrointestinal mucosa of a subject.

In recent years, the number of patients developing aortic valve stenosis has been increasing due to the aging population. Severe aortic valve stenosis is known to be associated with gastrointestinal bleeding, a condition known as Hyde syndrome. Hyde syndrome has been reported to be a condition in which the aortic valve narrows, and as blood passes through this narrowed area, high-molecular-weight von Willebrand factor (vWF) polymers, essential for hemostasis, are mechanically destroyed, leading to increased bleeding (Non-Patent Literature 1). Furthermore,

When high shear forces are applied to circulating blood, the vWF polymer is subjected to physical stress, temporarily loosening its molecular structure. It is known that this loosening is then cleaved by ADAMTS13, a specific zinc-containing metalloprotease. Therefore, the molecular size of the polymerized vWF is initially reduced (Non-Patent Literature 2). Patent Literature 1 and Non-Patent Literature 2 use the relative abundance of vWF polymers in the subject's blood sample as an indicator of the amount of vWF polymers. The retention rate of vWF polymers is measured by quantifying the relative abundance of vWF polymers in the subject's blood sample as a vWF polymer index, obtained by dividing the relative abundance of vWF polymers in the subject's blood sample by the relative abundance of vWF polymers in the blood sample of a healthy individual.

Japanese Patent Publication No. 2019-60772

Joseph Loscalzo, N ENGL J MED 367;20 nejm.org november 15, 2012 Hisanori Horiuchi et al., J Atheroscler Thromb, 2019; 26: 303-314. http://doi.org/10.5551/jat.RV17031

Gastrointestinal bleeding due to vascular dysplasia in the digestive tract can only be detected by endoscopy, so even if a patient experiences gastrointestinal bleeding, it is often treated as anemia of unknown cause. Furthermore, the extent of vascular dysplasia causing the bleeding can only be determined by endoscopy. However, endoscopy has its drawbacks, such as being a significant burden on the patient and being unsuitable for patients with dementia.
Furthermore, Non-Patent Document 2 states that the causal relationship between the development of vascular dysplasia and vWF polymer multimers is unclear.
The present invention aims to measure the incidence level of vascular dysplasia occurring in the digestive tract without performing an endoscopic examination.

Through diligent research, the inventors discovered a correlation between the level of vascular dysplasia occurring in the gastrointestinal mucosa and the amount of vWF high molecular weight polymers present in blood samples taken from patients with vascular dysplasia.
The present invention is completed based on the aforementioned findings and includes the following embodiments.
Item 1. A method for assisting in determining the level of vascular dysplasia in the gastrointestinal mucosa of a subject, comprising obtaining a subject's measurement data, which is the ratio or amount of high molecular weight von Willebrand factor (vWF) in the subject's blood sample, and a subject's vWF high molecular weight index determined based on a predetermined reference value corresponding to the measurement data, wherein the subject's vWF high molecular weight index reflects the level of vascular dysplasia in the gastrointestinal mucosa.
Item 2. The method according to Item 1, wherein the subject's vWF high molecular weight macromer index is calculated by adding, subtracting, multiplying, or dividing the subject's measurement data by a predetermined reference value. Item 3. The method according to Item 1 or 2, wherein the predetermined reference value is determined based on measurement data of high molecular weight macromers of von Willebrand factor (vWF) in blood samples from healthy individuals.
Item 4. The method according to any one of items 1 to 3, further comprising applying the obtained vWF high molecular weight macromer index of the subject to a calibration curve created based on vWF high molecular weight macromer indices obtained from at least two blood samples taken from control subjects with different levels of vascular dysplasia in the gastrointestinal mucosa, and presenting the level of vascular dysplasia in the subject determined from the calibration curve.
Item 5. The method according to any one of items 1 to 4, wherein the occurrence level is presented semi-quantitatively.
Item 6. The method according to any one of items 1 to 4, wherein the occurrence level is quantitatively presented.
Item 7. The method according to any one of items 1 to 6, wherein the subject is a patient suspected of having acquired von Willebrand syndrome.
Item 8. A device for assisting in determining the level of vascular dysplasia in the gastrointestinal mucosa of a subject, the device comprising a processing unit, the processing unit including obtaining a subject's vWF high molecular weight index determined based on the subject's measurement data, which is the ratio or amount of high molecular weight von Willebrand factor (vWF) in the subject's blood sample, and a predetermined reference value corresponding to the measurement data, wherein the subject's vWF high molecular weight index reflects the level of vascular dysplasia in the gastrointestinal mucosa.
Item 9. A computer program for assisting in determining the level of vascular dysplasia in the gastrointestinal mucosa of a subject, comprising the step of causing the computer to perform the steps of obtaining the subject's measurement data, which is the ratio or amount of high molecular weight von Willebrand factor (vWF) in the subject's blood sample, and the subject's vWF high molecular weight index determined based on predetermined reference values corresponding to the measurement data, wherein the subject's vWF high molecular weight index reflects the level of vascular dysplasia in the gastrointestinal mucosa.
Item 10. A test reagent for use in the method described in any one of items 1 to 7, comprising an antibody for detecting von Willebrand factor (vWF).
Item 11. A test kit for use in the method described in any one of items 1 to 7, comprising the test reagent described in item 10.
Item 12. A sample buffer reagent for use in the method described in any one of items 1 to 7, comprising a buffer, a chelating agent, and a surfactant.

According to the present invention, it is possible to predict the level of vascular dysplasia occurring in the patient's gastrointestinal mucosa without performing an endoscopic examination.

The hardware configuration of device 10 is shown. The processing flow of the decision assistance program 1042 is shown below. This shows the number (n) and percentage of gastrointestinal vascular lesions detected in complete gastrointestinal endoscopic examinations. The results of Western blotting for each patient, along with quantitative results (Small Multimer, Medium Multimer, Large Multimer), the numerical value calculated using Equation 5 (Large Multimer Ratio), the vWF high molecular weight macromer index (Large Multimer Index (%)) obtained by multiplying it by 100, and the number of gastrointestinal bleeding disorders for each patient are shown. In each lane, "Control" represents the negative control, and the numbers indicate the identification number of each patient. The results of Western blotting for each patient, along with quantitative results (Small Multimer, Medium Multimer, Large Multimer), the numerical value calculated using Equation 5 (Large Multimer Ratio), the vWF high molecular weight macromer index (Large Multimer Index (%)) obtained by multiplying it by 100, and the number of gastrointestinal bleeding disorders for each patient are shown. In each lane, "Control" represents the negative control, and the numbers indicate the identification number of each patient. This shows the correlation between the number of small intestinal gastrointestinal mucosal angioplasia lesions and the vWF high molecular weight macromer index. Endoscopic images of the small intestine before and after aortic valve replacement surgery are shown. (A) is the image before surgery, and (B) is the image after surgery.

1. Method for Assisting in Determining the Level of Angiodysplasia The present invention relates to a method for assisting in determining the level of angiodysplasia in the gastrointestinal mucosa of a subject. The method includes obtaining the subject's vWF high molecular weight index, which is determined based on the subject's measurement data, which is the ratio of high molecular weight von Willebrand factor (vWF) polymers in the subject's blood sample, and predetermined reference values. The subject's vWF high molecular weight index reflects the level of angiodysplasia in the gastrointestinal mucosa.

vWF is a type of coagulation factor whose monomer consists of 2050 amino acid residues. In the blood, vWF forms multimers ranging from 2-mer to approximately 80-mer in length, and functions to aggregate platelets at the bleeding site when bleeding occurs. This function is considered to be stronger as the vWF multimer becomes higher in length. Therefore, not only is the presence of vWF within a normal range concentration in the blood important, but the ratio of high-molecular-weight vWF multimers (hereinafter sometimes referred to as "vWF high-molecular-weight multimers") is also important for a normal primary hemostatic response, including platelet aggregation at the bleeding site. vWF is considered a low-molecular-weight multimer from 2-mer to 10-mer, a medium-molecular-weight multimer from 12-mer to 20-mer, and a high-molecular-weight multimer of 22-mer or more. Since vWF is based on dimers, when vWF molecules bind to each other, they bind in pairs of 2-mer.

In this specification, the digestive tract may include the stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum, ascending colon, transverse colon, descending colon, sigmoid colon, rectum), and esophagus. Preferably, the digestive tract consists of the small intestine, stomach, and large intestine, and more preferably, the small intestine. In the small intestine, when the small intestine is divided into three sections from below the pylorus—the upper, middle, and lower small intestines—the incidence of vascular dysplasia decreases in the order of the upper, middle, and lower small intestines. Furthermore, in the large intestine, when the section from the cecum to the sigmoid colon is divided into the right colon (cecum, ascending colon, transverse colon) and the left colon (descending colon, sigmoid colon), the frequency of vascular dysplasia is higher in the right colon than in the left colon and rectum.

In this specification, vascular dysplasia may include at least one of the following three lesions: i. Lesions with venous characteristics: Lesions in which abnormal blood vessels with venous characteristics, lacking an internal elastic lamina consisting of a thin vessel wall, are dilated and tortuous.
ii. Lesions with arterial characteristics: These are lesions in which abnormally large arteries, which are not normally present in the submucosa, are tortuous and located close to the mucosa.
iii. Lesions exhibiting characteristics of both arteries and veins: These are lesions that have an anastomosis or transition between relatively large arteries and veins.

The subjects are not particularly limited. Preferably, they are individuals suspected of having high shear stress on their circulating blood in a part of their circulatory system. Diseases that may cause high shear stress include cardiac valve stenosis (aortic valve stenosis, mitral valve stenosis, etc.), cardiac valve regurgitation (aortic valve regurgitation, mitral valve regurgitation, etc.), valvular heart disease (bacterial endocarditis, viral endocarditis, etc.), and left ventricular hypertrophy. Individuals with artificial hearts (implantable ventricular assist devices, extracorporeal artificial hearts, etc.) or artificial cardiovascular systems may also be subjects, as high shear stress may be placed on their circulating blood. Furthermore, individuals who are not suspected of having high shear stress on their circulating blood may also be subjects.

Those suspected of having high shear stress on their circulating blood are, in other words, patients suspected of having acquired von Willebrand syndrome. Von Willebrand syndrome has three types: type 1, type 2, and type 3. Acquired von Willebrand syndrome is classified as type 2.

A blood sample is blood collected from a subject, or a sample obtained by processing collected blood. Examples of blood samples include whole blood and plasma. It is preferable to collect blood using an anticoagulant other than heparin preparations. Citrate, such as sodium citrate solution, can be used as the anticoagulant. For example, plasma separated from a blood sample prepared by mixing a 3.1% to 3.3% (weight/volume) trisodium citrate solution with collected blood in a volume ratio of approximately 1:8.5 to 1:9.5 can be used as a blood sample.

The measurement data indicates the amount or ratio of high-molecular-weight vWF polymers in the blood sample. Methods for measuring the molecular size of vWF polymers have already been published in Patent Document 1 and Non-Patent Document 2. Briefly, for example, a blood sample is applied to a 1% agarose gel, and electrophoresis is performed at 3 mA for 10-12 hours or at 2 mA for approximately 20 hours.

The buffer for preparing the agarose gel may contain, for example, 3.0 g of Tris, 15.0 g of Glycine, 0.5 g of SDS, and 150 ml of Glycerol in 500 ml. The electrophoresis buffer may contain 6.0 g of Tris, 30.0 g of Glycine, and 1.0 g of SDS in 1000 ml. It is preferable to dilute the blood sample approximately 25-fold with the sample buffer. 50 ml may contain 174 μl of 3M Tris (pH 6.8), 208 μl of 0.5M EDTA, 5 ml of 20% SDS, 1 ml of 1% BPB, and 43.6 ml of 50% Glycerol. After mixing the blood sample with the sample buffer, it should be heated at approximately 56°C for about 20 minutes, and then immediately cooled on ice for about 3 minutes. If there is time before analysis, freeze the mixture of blood sample and sample buffer at approximately -80°C after ice cooling.

After electrophoresis, proteins are transferred from the agarose gel to a PVDF membrane using standard Western blotting techniques. The vWF on the PVDF membrane is then detected as a signal using an anti-human vWF antibody (e.g., polyclonal rabbit anti-human vWF antibody; DAKO).

When using an anti-human vWF antibody as the primary antibody, vWF is visualized by labeling the secondary antibody corresponding to the primary antibody with a labeling substance and obtaining a signal from the labeling substance. The labeling substance is not particularly limited as long as a detectable signal is produced. Examples include enzymes, fluorescent substances, radioisotopes, and metal nanoparticles. Examples of enzymes include peroxidase and alkaline phosphatase. Examples of fluorescent substances include fluorescent dyes such as fluorescein isothiocyanate (FITC), rhodamine, and Alexa Fluor®, and fluorescent proteins such as GFP. Examples of radioisotopes include ^125I , ^14C , and ^32P . Examples of metal nanoparticles include gold nanoparticles and silver nanoparticles. Among these, peroxidase is preferred as the labeling substance. Labeled antibodies may be purchased commercially. Alternatively, the labeling substance may be directly labeled onto the primary antibody. Labeling can be performed using commercially available labeling kits.

A known method can be used to detect the signal. In this method, a measurement method can be appropriately selected according to the type of signal derived from the labeling substance mentioned above. For example, when the labeling substance is an enzyme, the signals such as light and color generated by reacting the substrate with the enzyme can be visualized using an image analyzer (e.g., LAS4000 (GE Healthcare)). The enzyme substrate can be appropriately selected from known substrates depending on the type of enzyme. For example, when using alkaline phosphatase as the enzyme, chemiluminescent substrates such as CDP-Star® (4-chloro-3-(methoxyspiro[1,2-dioxetane-3,2'-(5'-chloro)trixyl[3.3.1.13,7]decane]-4-yl)phenyl phosphate disodium) can be used as substrates. When the labeling substance is a peroxidase, examples include the ImmunoStar® series (Fujifilm Wako Pure Chemical Corporation), Pierce ECL (Thermo Fisher Scientific K.K.), Pierce ECL Plus (Thermo Fisher Scientific K.K.), and West Dura (Thermo Fisher Scientific K.K.).

If the labeling material is a radioactive isotope, the radiation signal can be measured using known instruments such as autoradiography or scintillation counters. If the labeling material is a fluorescent material, the fluorescence signal can be measured using known instruments such as image analyzers. The excitation and fluorescence wavelengths can be appropriately determined depending on the type of fluorescent material used. If the labeling material is a metal nanoparticle, the color change due to the aggregation of the metal nanoparticles can be measured using known instruments such as image analyzers.

Western blotting causes vWF molecules to arrange themselves in a ladder-like structure according to their molecular size. The smallest molecular size bands are dimers, and they are arranged in a ladder-like structure with two molecules per band, moving towards the higher molecular size side. Low-molecular-weight vWF macromers correspond to the first to fifth ladders, counting from the low molecular size side. Medium-molecular-weight vWF macromers correspond to the sixth to tenth ladders, counting from the low molecular size side. High-molecular-weight vWF macromers correspond to the eleventh and above ladders, counting from the low molecular size side. Therefore, by quantifying the signal intensity of these ladders using an image analyzer or similar device, the abundance or ratio of high-molecular-weight vWF macromers in a blood sample can be obtained.

Furthermore, the subject's vWF high molecular weight polymer index can be obtained based on measurement data acquired from the subject's blood sample and the corresponding predetermined reference values.

The predetermined reference values can be determined based on measurement data obtained from blood samples taken from individuals whose circulating blood is not subjected to high shear stress and who have no abnormalities in other clinical test data, including blood coagulation function (hereinafter referred to as "healthy individuals" for convenience). Preferably, the blood samples used to determine the reference values are taken from multiple different healthy individuals. The reference values may be the measurement data of healthy individuals themselves, the average value of measurement data obtained from multiple healthy individuals, the value obtained by adding the standard deviation (or twice the standard deviation) to the average value, the value obtained by subtracting the standard deviation (or twice the standard deviation) from the average value, the median, the first quartile, the third quartile, etc.

Corresponding to the subject's measurement data means that when the subject's measurement data represents an abundance, the reference value is also an abundance; and when the subject's measurement data represents an abundance ratio, the reference value is also an abundance ratio.

The subject's vWF high molecular weight polymer index can be calculated by adding, subtracting, multiplying, or dividing the subject's measurement data by a predetermined reference value. More specifically, it can be calculated using one of the following formulas.
vWF high molecular weight polymer index = (measurement data of the subject) + (reference value) (Equation 1)
vWF high molecular weight polymer index = (measurement data of the subject) - (reference value) (Equation 2)
vWF high molecular weight polymer index = (reference value) - (measurement data of the subject) (Equation 3)
vWF high molecular weight polymer index = (measurement data of the subject) × (reference value) (Equation 4)
vWF high molecular weight polymer index = (measurement data of the subject) ÷ (reference value) (Equation 5)
vWF high molecular weight polymer index = (reference value) ÷ (measurement data of the subject) (Equation 6)
In the above formulas, "+" indicates addition, "-" indicates subtraction, "×" indicates multiplication, and "÷" indicates addition.

The subject's vWF high molecular weight polymer index reflects the level of vascular dysplasia in the gastrointestinal mucosa. The level of vascular dysplasia refers to, for example, the number of vascular dysplasia lesions occurring in a given area of the gastrointestinal mucosa, or the area of vascular dysplasia occurring in a given area of the gastrointestinal mucosa. The area may include the area of a single location or the sum of the areas of multiple locations. Specifically, in equations 1, 2, 4, and 5, a smaller vWF high molecular weight polymer index indicates a larger number of vascular dysplasia lesions or a larger area of vascular dysplasia in the gastrointestinal mucosa. Conversely, in equations 1, 2, 4, and 5, a larger vWF high molecular weight polymer index indicates a smaller number of vascular dysplasia lesions or a smaller area of vascular dysplasia in the gastrointestinal mucosa. On the other hand, in equations 3 and 6, a smaller vWF high molecular weight polymer index indicates a smaller number of vascular dysplasias occurring in the gastrointestinal mucosa or a smaller area of vascular dysplasia. Conversely, in equations 3 and 6, a larger vWF high molecular weight polymer index indicates a larger number of vascular dysplasias occurring in the gastrointestinal mucosa or a larger area of vascular dysplasia.

The subject's vWF polymer index is preferably calculated according to formula 5 or formula 6, and more preferably according to formula 5. Alternatively, the value obtained by formula 5 or formula 6 may be multiplied by 100 to express the vWF polymer index as a percentage (%).

Obtaining the vWF polymer index may involve calculating it according to the above formula, or obtaining the calculated vWF polymer index from another computer, facility, or institution.

Furthermore, the acquired vWF high molecular weight polymer index of the subject can indicate the level of vascular dysplasia in the subject's gastrointestinal mucosa. In this case, a calibration curve is created to convert the vWF high molecular weight polymer index to the level of vascular dysplasia. The calibration curve is created based on vWF high molecular weight polymer indices obtained from at least two blood samples taken from control subjects with different levels of vascular dysplasia in the gastrointestinal mucosa.

The control subjects are those referred to as the "golden standard," who have been determined to have vascular dysplasia in the gastrointestinal mucosa through actual imaging diagnostics such as endoscopy. The vWF high molecular weight macromer index obtained from blood samples taken from the control subjects can be linked to the actual level of vascular dysplasia occurring in the gastrointestinal mucosa. Therefore, the calibration curve is created based on the control subjects' blood samples and the level of vascular dysplasia in the gastrointestinal mucosa at the time the blood samples were taken. In this case, the control subjects may be the same person or different people. They may also be a mixture of different individuals. If the control subjects are the same person, for example, in the case of untreated vascular dysplasia in the gastrointestinal mucosa, blood samples are taken and information on the level of vascular dysplasia is obtained at multiple points in time during the process of treatment and improvement of the untreated vascular dysplasia. The collection of blood samples and the acquisition of information on the level of vascular dysplasia do not necessarily have to be on the same day, but it is preferable to do so within 3 days, preferably within 2 days, and ideally within 1 day.

The number of combinations of vWF high molecular weight polymer index and information on the incidence level of gastrointestinal mucosal vascular dysplasia necessary to generate a calibration curve is not limited as long as there are at least two. For example, preferably three or more, more preferably five or more. The greater the number of combinations of vWF high molecular weight polymer index and information on the incidence level of gastrointestinal mucosal vascular dysplasia, the more accurate the calibration curve becomes. The calibration curve may be a straight line or a curve. Since the calibration curve is a regression equation, the incidence level of gastrointestinal mucosal vascular dysplasia in the subject can be quantitatively determined by fitting the subject's vWF high molecular weight polymer index to the calibration curve.
The results indicating the incidence level of vascular dysplasia in the gastrointestinal mucosa may be quantitatively expressed as the numerical value itself calculated by the regression equation.

Furthermore, the results indicating the incidence level of gastrointestinal mucosal vascular dysplasia may be expressed semi-quantitatively, such as "high,""moderate," or "low." In this case, for example, the vWF high molecular weight polymer index is pre-defined within a certain numerical range according to the incidence level of gastrointestinal mucosal vascular dysplasia (e.g., three quantiles: "high,""moderate," and "low"). For each subject, the quantile corresponding to the numerical value output from the regression equation is presented as the result indicating the incidence level of gastrointestinal mucosal vascular dysplasia.
"Presentation" may include displaying on a screen such as a display, or displaying on paper or other media showing test results.

2. Apparatus for assisting in determining the level of vascular dysplasia (1) Hardware configuration One embodiment of the present invention is an apparatus 10 (hereinafter also referred to as "apparatus 10") for assisting in determining the level of vascular dysplasia in the gastrointestinal mucosa of a subject.

The hardware configuration of device 10 will be explained using Figure 1. Device 10 may be a general-purpose computer. Device 10 is communicated with an input device 111, an output device 112, and a media drive 113. Device 10 includes a CPU 101, memory 102, ROM (read-only memory) 103, storage device 104, communication interface (I/F) 105, input interface (I/F) 106, output interface (I/F) 107, and media interface (I/F) 108. Each component within device 10 is connected to each other via a bus 109 for data communication.

The storage device 104 is composed of a hard disk, a semiconductor memory element such as flash memory, an optical disc, etc. The storage device 104 stores an operating system (OS) 1041, a decision assistance program 1042 (described later), a reference value database (DB) DB1, and a calibration curve database (DB) DB3. The decision assistance program 1042 works in cooperation with the operating system 1041 to make the computer function as device 10. The reference value database DB1 stores predetermined reference values. The calibration curve database DB3 records information on the vWF high molecular weight polymer index of the control subjects and the incidence level of vascular dysplasia of the gastrointestinal mucosa, as well as regression equations calculated based on these. Furthermore, the calibration curve database DB3 may also store the numerical range of the quantized vWF high molecular weight polymer index, along with labels indicating the semi-quantitative results of the incidence level of vascular dysplasia of the gastrointestinal mucosa corresponding to each numerical range.
In this embodiment, the CPU 101 is also called the processing unit 101.
The reference range database DB1 stores the reference ranges described in 1-1 above.

The input device 111 consists of a touch panel, keyboard, mouse, pen tablet, microphone, etc., and performs text input or voice input to the device 10. The input device 111 may be connected externally to the processing unit 101 or may be integrated with the device 10.

The output device 112 consists of, for example, a display device such as a display, a printer, etc., and outputs various operation windows, results indicating the occurrence level of vascular dysplasia of the gastrointestinal mucosa, etc. The media drive 113 may be a USB drive, a flexible disk drive, a CD-ROM drive, or a DVD-ROM drive, etc. The communication interface 105 receives a group of raw data from the measuring device 50. The output interface 107 transmits the results to the output device 112.
(2) Processing flow of the decision assistance program Figure 2 shows the processing flow of the decision assistance program 1042.

In step S1, the processing unit 101 of the device 10 receives the measurement data of the subject input by the operator via the input device 111. It also receives a request for reference value acquisition from the operator via the input device 111 and acquires a predetermined reference value corresponding to the subject's measurement data from the reference value database DB1.

In step S2, the processing unit 101 receives an index calculation request input by the operator from the input device 111 and obtains the vWF polymer index according to the formula described in section 1 above.

In step S3, the processing unit 101 receives a request from the operator via the input device 111 to indicate the level of vascular dysplasia. If the received request quantitatively indicates the level of vascular dysplasia ("YES"), the processing unit 101 proceeds to step S4, retrieves a calibration curve from the calibration curve database DB3, applies the subject's vWF high molecular weight polymer index obtained in step S2 to the calibration curve, and generates a result indicating the level of vascular dysplasia. The processing unit 101 then proceeds to step S5 and outputs the result generated in step S4.

In step S3, if the received presentation request does not quantitatively present the level of vascular dysplasia ("NO"), the processing unit 101 proceeds to step S6 to determine whether it presents the level of vascular dysplasia semi-quantitatively. If the presentation request for the level of vascular dysplasia presents the level of vascular dysplasia semi-quantitatively ("YES"), the processing unit 101 proceeds to step S7. In step S7, the processing unit 101 obtains the quantile label corresponding to the subject's vWF high molecular weight polymer index obtained in step S2 from the calibration curve database DB3. The processing unit 101 then proceeds to step S5 and outputs the result obtained in step S7.

In step S6, if the request for presentation of the level of vascular dysplasia does not present the level of vascular dysplasia semi-quantitatively (i.e., "NO"), the processing unit 101 proceeds to step S8 and outputs the subject's vWF high molecular weight polymer index.

(3) Recording medium on which the decision assistance program is recorded The decision assistance program 1042, which comprises the processing of steps S1 to S8, may be recorded on a recording medium.

In other words, the decision-making assistance program 1042 is stored on a recording medium such as a hard disk, a semiconductor memory element such as flash memory, or an optical disc. The computer program may also be stored on a network-connectable recording medium such as a cloud server. The computer program may be provided as a downloadable program product or as a program product recorded on a recording medium.

The format in which the program is stored on the recording medium is not limited, as long as the device 10 can read the program. Storage on the recording medium is preferably non-volatile.

3. Test reagents and test kits One embodiment of the present invention relates to test reagents and test kits for performing a method to assist in determining the level of vascular dysplasia described in 1. above.

The test reagent includes one or more anti-vWF antibodies (e.g., primary antibodies) capable of binding to at least a portion of vWF. The "antibody" can be a polyclonal antibody, a monoclonal antibody, or a fragment thereof (e.g., Fab, F(ab'), F(ab) ₂ , etc.). The immunoglobulin class and subclass of the antibody are not particularly limited. Furthermore, the antibody may be screened from an antibody library, or it may be a chimeric antibody, scFv, etc. Also, commercially available antibodies as described in section 1 above may be used as the anti-vWF antibody.

Furthermore, the antibodies do not necessarily need to be purified; they may be antiserum containing antibodies, ascites fluid, or immunoglobulin fractions derived from these.

The antibodies contained in the test reagent may be in a dry state or dissolved in a buffer such as phosphate-buffered saline. Furthermore, the test reagent may contain at least one of the following: stabilizers such as β-mercaptoethanol and DTT; protective agents such as albumin; surfactants such as polyoxyethylene (20) sorbitan monolaurate and polyoxyethylene (10) octylphenyl ether; and preservatives such as sodium azide.
The antibody that binds to vWF may be labeled with a labeling substance. The labeling substance is preferably an enzyme, and more preferably a peroxidase.

The vWF detection reagent may be provided as a test kit containing an accompanying document describing the reagent and its usage, or a URL to a webpage describing its usage. Furthermore, if the antibody binding to vWF is an unlabeled primary antibody, the test kit may include a secondary antibody labeled with a labeling substance (preferably an enzyme, more preferably a peroxidase). The test kit may also include a substrate that reacts with the enzyme. Additionally, a sample buffer for mixing with the blood sample may be included in the kit.

4. Sample Buffer Reagent for Mixing with Blood Samples One embodiment of the present invention relates to a sample buffer reagent for mixing with blood samples. The sample buffer reagent is used to carry out the method for assisting in determining the level of vascular dysplasia described in 1. above. The sample buffer reagent may include a buffer based on Tris, etc., a chelating agent such as EDTA, a surfactant such as sodium dodecyl sulfate (SDS), etc., as described in 1. above. Furthermore, the sample buffer reagent may also include an electrophoretic marker dye such as bromophenol blue (BPB), and a specific gravity increaser such as glycerol, etc. The sample buffer reagent may be packaged in single-use portions in one container, or multiple portions may be stored in one container.

The present invention will be described in more detail below with reference to examples. However, the present invention is not to be interpreted as being limited to these examples.

After obtaining informed consent from 38 patients with severe aortic stenosis who visited Kyoto Prefectural University of Medicine, a complete gastrointestinal endoscopic examination (esophagus to colon) and quantitative measurement of vWF high molecular weight polymers were performed.
The high molecular weight vWF polymer was quantified by Western blotting. A 1% agarose gel was used for electrophoresis, and a PVDF membrane was used. Polyclonal rabbit anti-human vWF antibody/HRP (Dako) was used as the antibody to detect vWF. An immunostaseta (Fujifilm Wako Pure Chemical Industries, Ltd.) was used for detection, and the Image J system was used for band quantification.

All patients included in the study had hemoglobin levels lower than 11 g/dl. Figure 3 shows the number and percentage (%) of gastrointestinal mucosal vascular lesions in all cases. Vascular lesions (vascular dysplasia) were observed in at least the gastrointestinal mucosa of the stomach, small intestine, and large intestine in all cases. In particular, vascular lesions were observed in 90% of cases in the small intestine.

Figures 4(A) and (B) show the results of Western blotting for each patient, along with quantitative results (Small Multimer, Medium Multimer, Large Multimer), the numerical value calculated using Equation 5 (Large Multimer Ratio), the vWF high molecular weight multimer index (Large Multimer Index (%)) obtained by multiplying the Large Multimer Ratio by 100, and the number of gastrointestinal bleeding lesions for each patient. In each lane, "Control" represents the negative control, and the numbers indicate the identification number of each patient. The number of lesions was counted as the number of locations where vascular dysplasia lesions were observed throughout the entire small intestine (upper, middle, and lower intestines). Figure 4(C) shows the correlation between the vWF high molecular weight multimer index and the number of gastrointestinal mucosal vascular dysplasia lesions (number of lesions). A tendency was observed for the vWF high molecular weight multimer index to be lower as the number of lesions increased. This indicates that the vWF high molecular weight multimer index correlates with the level of vascular dysplasia in the gastrointestinal mucosa.

Figure 5 shows endoscopic images of the small intestine before and after aortic valve replacement surgery. (A) is the pre-operative image, and (B) is the post-operative image. Before surgery, the circulating blood was subjected to high shear stress, resulting in a wide area of vascular lesions in the small intestine. However, after aortic valve replacement surgery, the high shear stress was eliminated, and the vascular lesions improved.

10 Device 101 Processing Unit 1042 Decision Assistance Program

Claims (11)

Obtain the subject's measurement data, which is the amount of high molecular weight von Willebrand factor (vWF) polymer present in the subject's blood sample, and the subject's vWF high molecular weight polymer index, which is determined based on predetermined reference values corresponding to the measurement data .
This includes applying the obtained vWF high molecular weight polymer index of the subject to a calibration curve created based on vWF high molecular weight polymer indices obtained from at least two blood samples taken from control subjects with different levels of vascular dysplasia in the small intestinal mucosa, and presenting the level of vascular dysplasia in the subject determined from the calibration curve .
A method to assist in determining the level of vascular dysplasia in the small intestinal mucosa of a subject. The method according to claim 1, wherein the subject's vWF polymer index is calculated by adding, subtracting, multiplying, or dividing the subject's measurement data by a predetermined reference value. The method according to claim 1 or 2, wherein a predetermined reference value is determined based on measurement data of high molecular weight von Willebrand factor (vWF) in blood samples from healthy individuals. The method according to claim 1 , wherein the occurrence level is presented semi-quantitatively. The method according to claim 1 , wherein the occurrence level is presented quantitatively. The method according to claim 1 , wherein the subject is a patient suspected of having acquired von Willebrand syndrome. A device for assisting in determining the level of vascular dysplasia in the small intestinal mucosa of a subject,
The device comprises a processing unit,
The aforementioned processing unit,
The subject's measurement data, which is the amount of high molecular weight von Willebrand factor (vWF) polymer present in the subject's blood sample, and the subject's vWF high molecular weight polymer index, determined based on predetermined reference values corresponding to the measurement data, are obtained.
The obtained vWF high molecular weight macromer index of the subject is applied to a calibration curve created based on vWF high molecular weight macromer indices obtained from at least two blood samples taken from control subjects with different levels of vascular dysplasia in the small intestinal mucosa. The level of vascular dysplasia in the subject's small intestinal mucosa, determined from the calibration curve, is then presented .
The aforementioned device. When you run it on a computer, the computer will
The steps include obtaining the subject's measurement data, which is the amount of high molecular weight von Willebrand factor (vWF) polymer present in the subject's blood sample, and the subject's vWF high molecular weight index determined based on predetermined reference values corresponding to the measurement data ,
The obtained vWF high molecular weight macromer index of the subject is applied to a calibration curve created based on vWF high molecular weight macromer indices obtained from at least two blood samples taken from control subjects with different levels of vascular dysplasia in the small intestinal mucosa, and the level of vascular dysplasia in the subject's small intestinal mucosa determined from the calibration curve is presented .
A computer program to assist in determining the level of vascular dysplasia in the small intestinal mucosa of a subject. A test reagent for use in the method according to claim 1 , comprising an antibody for detecting von Willebrand factor (vWF). A test kit for use in the method according to claim 1 , comprising the test reagent described in claim 9 . A sample buffer reagent for use in the method according to claim 1, comprising a buffer, a chelating agent, and a surfactant.